3.2.1 Fruit used for isolation of potential antagonists
Fruit of papaya (Carica papaya L.), granadilla (Passiflora quadrangularis L.) and a range of citrus [i.e., navel orange (Citrus sinensis [L] Osbeck), Valencia orange (Citrus sinensis [L] Osbeck), lemon (Citrus limon Burmann), grapefruit (Citrus paradisi Macf) and mandarin (Citrus reticulata Blanco)] were harvested from commercial orchards and home guardens in KwaZulu-Natal and Mpumalanga, South Africa. Undamaged fruit were processed either immediately, after storage for 2-3 days at room temperature or after storage in a cold room at 8±1°C for 5-7 days.
3.2.2 Preliminary investigation of microorganisms on the fruit surface Navel and Valencia oranges that had not been sprayed with fungicides were collected from citrus orchards located at Ukulinga Research Farm, University of KwaZulu-Natal, Pietermartizburg (29.36 S 30.24 E), South Africa, and stored at 25°C for two weeks. Whole oranges were rinsed with sterile water in order to remove any potential antagonists. The rinsing water was then serially diluted (10 fold dilution series was made up to 10-4) and plated onto potato dextrose agar (PDA) (Merck Laboratory, South Africa). The plates were incubated at 25°C for four days. Yeasts or/and Bacillus colonies were selected and identified visually by their typical colony morphologies.
3.2.3 Isolation of antagonistic yeasts and Bacillus
Bacillus and yeast isolates were recovered from the peel of 3-5 mature fruit from a range of the fruit samples, as described in Section 3.2.2. The fruit peel was cut into 10-15 small pieces, weighing 50 grams and placed in separate 250 mℓ Erlenmeyer flasks containing 100 mℓ sterile distilled water plus quarter strength Ringer‟s Solution and shaken in a water bath (G.F.L. 1083, Labortechnik, Germany) at 120 rotations per minute (rpm) for one hour at 30°C.
Fruit peel pieces were removed and the liquid suspension was used to make a serial dilution of 10-1, 10-2, 10-3 and 10-4. An aliquot of 0.2 mℓ of each dilution was plated onto PDA amended with 0.15 g ℓ-1 of Rose Bengal (BDN Laboratory, England) for recovery of yeast isolates and incubated at 25°C for three days.
Pure cultures of yeast were made by sub-culturing from discrete colonies on the plates. For isolation of Bacillus, the same serial dilution prepared for the yeast isolates was used, after heat treatment at 80°C for 15 minutes in a water bath.
Each aliquot of 0.2 mℓ was poured onto a tryptone soy agar (TSA) (Merck Laboratory, South Africa) plate. Plates were incubated for 3 days at 28°C, after which representative colonies were arbitrarily selected and streaked onto fresh TSA plates to obtain single colonies. Isolates were stored in sterile distilled water and -80°C freeze in 20% glycerol.
3.2.4 Screening of yeasts and Bacillus isolates against P. digitatum 3.2.4.1 Preliminary Screening
A total of 60 yeast and 92 Bacillus isolates (see Section 3.2.1) were tested on navel oranges. Each fruit was surface disinfected with 70% alcohol for
disinfected needle 10 times at both ends of the fruit. The wound was dipped into a suspension of yeast cells or Bacillus (1 × 108 cells mℓ-1) for one minute. Three hours after the wound site had dried, each wounded fruit was dipped into a suspension of conidia of P. digitatum at 1 × 104 conidia mℓ-1, isolated from infected navel oranges, collected at Gateway Packhouse (29.53 S 30.17 E), Thornville, Pietermaritzburg, South Africa. The conidial suspension was quantified using a haemocytometer and then adjusted to the final concentrations by dilution. Control fruit were treated with sterile distilled water.
Fruit were kept at room temperature (24±1°C) for 10 days. One box with three fruit was used per treatment. Treatments were placed on a bench. Fruit were examined for percentage fruit surface area covered by P. digitatum using visual estimations for the initial screening. The criterion used to select the antagonists was the ability to reduce growth or development of P. digitatum to
≤50%.
3.2.4.2 Secondry Screening
Further tests were conducted on the most promising yeast (10) and Bacillus (10) isolates (a detailed description of locations from where fruit has been obtained and isolates of yeast and Bacillus recovered is presented in Appendix 3A) following the procedures described in Section 3.2.4.1. However, in this instance Valencia oranges were used for the evaluation, and fruit was wounded (3 mm in length × 3 mm in depth) at one site on the fruit equator with a dissecting needle. The wound was then treated with a 100 μℓ cell suspensionof the test organism (yeast or Bacillus at 1 ×108 cells mℓ-1). After the wound site had dried for three hours, 100 μℓ of the conidial suspension of P. digitatum (1 × 104 conidia mℓ-1) was inoculated into the wound. Wounds inoculated with the same amount of P. digitatum isolate, but no biocontrol pretreatment, served as the control. Fruit were kept at room temperature (24±1°C). Two boxes, with five fruit per box, were used per treatment and placed on a bench in a complete randomized block design (CRBD). The criterion used to select the antagonist was the reduction of lesion diameters (mm) caused by P. digitatum 10 days after inoculation. Lesion diameter was measured by taking the mean of the horizontal and vertical diameters of each lesion.
3.2.5 A selected yeast and Bacillus isolates antagonistic against P.
digitatum
3.2.5.1 Preventative Action
Ten yeasts and 10 Bacillus isolates were tested for their preventative action against P. digitatum. This was achieved by treating fruit with an antagonist 48 hours before inoculation with the pathogen. The procedures described in Section 3.2.4.2 were followed for the preparation of the antagonist, P. digitatum, and for the treatment application. Navel and Valencia oranges, as wellas lemons were used in this trial. The fruit wound was extended to 25 mm in length
× 3 mm in depth. The wound was treated with 100 μℓ of cell suspension of the test organism (yeast or Bacillus at 1 × 108 cells mℓ-1). After the wound site had dried for 48 hours, each wound was inoculated with 100 μℓ of the suspension of conidia of the P. digitatum isolate (1 × 104 conidia mℓ-1). Wounds inoculated with the same amount of P. digitatum isolate served as the control. Fruit were kept at room temperature (24±1°C). Two boxes, with five fruit per box, were used per treatment and placed on a bench in a CRBD. Lesion diameter (mm) of each infected wound was determined 10 days after inoculation. Lesion diameter was measured by taking the mean of the horizontal and vertical diameters of each lesion.
3.2.5.2 Curative Action
Similar procedures as described in Section 3.2.5.1 were followed, with the difference that navel oranges were not included because they were out of season. Valencia oranges or lemons were wounded, and then inoculated with 100 μℓ of the suspension of P. digitatum conidia (1 × 104 conidia mℓ-1). After the wound site had dried for three hours, the wound was treated with a 100 μlof the test organism (yeast or Bacillus at 1 × 108 cells mℓ1). Wounds inoculated with P.
digitatum conidial suspension served as a control. Fruit were kept at room temperature (24±1°C). Two boxes, with five fruit per box, were used per treatment and placed on a bench in a CRBD. Lesion diameter was measured by taking the mean of the horizontal and vertical diameters of each lesion.
3.2.6 Dose effect of two yeast isolates, B13 and Grape, applied preventatively on lemons for the control of P. digitatum
The effect of various concentrations of two yeast isolates, namely, B13 and Grape (identified as strains of Candida fermentati (Saito) Bai. by Botes1, were studied for their preventative action on lemons against P. digitatum. Lemons were surface disinfected with 70% alcohol for 1 minute, dried, and then wounded. Fruit were wounded (25 mm in length × 3 mm in depth) at one site on the equator with a dissecting needle as described in Section 3.2.5.1. Cell suspensions (100 μℓ) of both yeasts of 1 × 105, 1 x 106, 2.5 × 106, 1 × 107 and 1 x 108cells mℓ-1 were inoculated into each wound site. The technique used was based on that reported by Tian et al. (2002). Wounds treated with 100 μℓ of distilled water served as a control. After 48 hours, all yeast-treated wounds and control wounds were inoculated with a 100 μℓ conidial suspension of P.
digitatum (1 × 104 conidia mℓ-1). Fruit were kept at room temperature (24±1°C).
Two boxes, with five fruit per box, were used per treatment and placed on a bench in a CRBD. Lesion diameter was measured by taking the mean of the horizontal and vertical diameters of each lesion.
3.2.7 Statistical analysis
With one exception, all data sets were analysed using a REML (REsidual Maximum Likelyhood) Variance Component Analysis using Genstat® Executable Release 9.1 Statistical Analysis Software (Anonymous, 2006).
Where the F test was significant, differences between treatment means were determined using Duncan‟s Multiple Range Test (P 0.05). Detailed analyses are presented in Appendixes 3B-3E.
The exception was in the case of measuring dose effects of two yeast isolates, B13 and Grape, for the control of P. digitatum, when applied preventatively on lemons. In this case, the data was subjected to an analysis of variance (ANOVA) using Genstat® Executable Release 9.1 Statistical Analysis Software (Anonymous, 2006). To determine differences between treatments, Fisher‟s Least Significant Difference Test was used (P<0.05).
1 Dr Botes, A. University of Stellenbosch, South Africa.